Bonev B et al. (2011), MicroRNA-9 reveals regional diversity of neural...

XB-ART-42850

Dev Cell 2011 Jan 18;201:19-32. doi: 10.1016/j.devcel.2010.11.018.

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MicroRNA-9 reveals regional diversity of neural progenitors along the anterior-posterior axis.

Bonev B , Pisco A , Papalopulu N .

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Neural progenitors self-renew and generate neurons throughout the central nervous system. Here, we uncover an unexpected regional specificity in the properties of neural progenitor cells, revealed by the function of a microRNA--miR-9. miR-9 is expressed in neural progenitors, and its knockdown results in an inhibition of neurogenesis along the anterior-posterior axis. However, the underlying mechanism differs--in the hindbrain, progenitors fail to exit the cell cycle, whereas in the forebrain they undergo apoptosis, counteracting the proliferative effect. Among several targets, we functionally identify hairy1 as a primary target of miR-9, regulated at the mRNA level. hairy1 mediates the effects of miR-9 on proliferation, through Fgf8 signaling in the forebrain and Wnt signaling in the hindbrain, but affects apoptosis only in the forebrain, via the p53 pathway. Our findings show a positional difference in the responsiveness of progenitors to miR-9 depletion, revealing an underlying divergence of their properties.

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Species referenced: Xenopus
Genes referenced: ccnd1 cdknx fgf8 hes1 mdm2 myt1 neurod1 rpl8 sox3 tp53 tubb2b wnt1 zic1
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	Figure 1. miR-9 Expression Differs along the AP Axis(A) Whole-mount in situ hybridization for miR-9 expression in X. tropicalis using LNA probe.(B) Expression of miR-9 primary transcripts at stage 30—dorsal view. Dashed line indicates the plane of sectioning in (D). MHB is indicated with an asterisk. Scale bar, 200 μm.(C) Schematic representation of the different regions in the neural tube (red, forebrain; green, midbrain; blue, hindbrain) and transverse sections from the forebrain and hindbrain (red, progenitors; purple, intermediate zone; blue, neurons).(D) In situ hybridization for miR-9 precursors in transverse sections from stage 30 embryos. CNS tissue is outlined with a dashed line. Scale bar, 20 μm.(E) FISH for miR-9a-1 (in red) combined with immunohistochemistry (IHC) for Sox3 (marker for neural progenitors) in stage 30 embryo. CNS tissue is outlined with a dashed line. DNA is stained with DAPI (4,6-diamidino-2-phenylindole). Scale bar, 20 μm.
	Figure 2. miR-9 Is Required for Neuronal Differentiation(A) Semiquantitative PCR analysis of mature miR-9 levels in stage 30 WT embryos, injected with miR-9-2 precursor or miR-9 MO at one cell stage. The snRNA U2 is used as a loading control.(B) Experimental outline. miR-9 MO was injected in one cell of the two-cell stage embryo, and the injected side was compared to the control at stage 30.(C) In situ hybridization (whole-mount and transverse sections from the forebrain and hindbrain) with markers for differentiated (N-tubulin) and differentiating neurons (NeuroD1). Note the reduced expression of both markers (arrows) in the miR-9 MO-injected side.(D) Immunohistochemistry on sections for the transcription factor Myt1 indicates impaired neuronal differentiation upon miR-9 knockdown. The FITC tag on miR-9 MO was used to identify the injected side; DAPI was used to stain the DNA.(E) The percentage of Myt1-positive cells in miR-9 MO-injected side relative to the control side in the forebrain (n = 6 embryos, p < 0.001) and hindbrain (n = 9 embryos, p < 0.001). Error bars represent SEM. In all images, scale bars represent 20 μm and CNS tissue is outlined with a dashed line.
	Figure 3. miR-9 Regulates Progenitor Proliferation in a Region-Specific Manner(A) Immunohistochemistry on sections for Sox3 shows expansion of the progenitor domain in the hindbrain.(B) Area occupied by Sox3-positive cells (progenitor domain) per section in miR-9 MO-injected side expressed relative to the control side in the forebrain (n = 7, p = 0.008) and hindbrain (n = 9, p < 0.001).(C and D) Transverse sections from the forebrain or hindbrain of miR-9 MO-injected embryos analyzed for the mitotic marker pH3 show a hindbrain-specific increase in the number of mitotic cells (n = 11, p < 0.001), but no change in the forebrain (n = 9).(E) pH3-labeling index (pH3+ cells over Sox3+ cells) in the hindbrain (n = 6, p = 0.004).(F and G) Rate of proliferation of the hindbrain progenitors is increased, as determined by BrdU incorporation for 30 min. BrdU-labeling index is calculated as the percentage of BrdU+ and Sox3+ cells over the total population of Sox3+ cells (n = 7, p < 0.001). In all panels, scale bars represent 20 μm, FITC staining shows the MO-injected side, DNA was counterstained with DAPI, CNS tissue is outlined with a dashed line, and error bars represent SEM.
	Figure 4. miR-9 Depletion Negatively Affects the Survival of Forebrain Neural Progenitors(A) TUNEL staining shows increased apoptosis upon miR-9 depletion in the forebrain (arrowheads), but not in the hindbrain.(B) Percentage of the TUNEL+ cells in the injected compared to the control side in the forebrain (n = 6, p < 0.001) and in the hindbrain (n = 6). Error bars represent SEM.(C and D) Sox3-positive domain is expanded in miR-9 MO-injected side when apoptosis is prevented (n = 7, p < 0.001). (E and F) The reduced number of differentiating neurons (Myt1+) upon miR-9 depletion is not rescued by caspase inhibitor block of apoptosis (n = 6, p = 0.003). In all images FITC staining shows the MO-injected side; DNA was counterstained with DAPI. Neural tube is outlined with a dashed line. Scale bars, 20 μm. Error bars represent SEM.
	Figure 5. miR-9 Regulates the Expression of hairy1 In Vivo(A) Sequence alignment of the predicted miR-9 binding site in HES1 homologs in human, mouse, Xenopus, and zebrafish. Positions that have a single, fully conserved residue are marked with an asterisk. Seed-complementary region is boxed in red.(B) HeLa cells were transfected with WT Xenopus hairy1 (xhairy1_WT), mouse Hes1 (mHes1), or mutant hairy1 (xHairy1_Mut) reporter together with either scrambled (Control) or miR-9 precursors (miR-9). Luciferase expression was normalized and expressed relative to the control levels. Error bars represent SD.(C) Design of target protector morpholino (Hairy1 TP) directed against hairy1 miR-9 binding site. Seed region is boxed in red.(D) Hairy1 TP alleviates the repression of hairy1 luciferase reporter when cotransfected with miR-9 precursors but has no effect on the repression of other miR-9 targets. Error bars represent SD.(E) In situ hybridization for miR-9 (miR-9a-1 transcript) and hairy1 in stage 30 embryos. Shown are whole mounts and transverse sections through the respective brain areas.(F) Double-fluorescent in situ for hairy1 (red) and miR-9a-1 (green) shows mutually exclusive pattern of expression along the AP axis.(G) miR-9 MO and hairy1 TP lead to expansion of the hairy1-positive domain (red arrowheads) along the AP axis, as shown by in situ hybridization.(H) Quantification of the change in hairy1 mRNA expression using qRT-PCR.(I) Hes1 mRNA levels in N1E neuroblastoma cells are downregulated when miR-9 is overexpressed and increased when it is knocked down using LNA inhibitors. In all graphs, data are presented as mean values, and error bars represent SEM.
	Figure 6. Hairy1 Target Protector Mimics miR-9 MO Phenotype(A) In situ hybridization (whole-mount and transverse sections from the forebrain and hindbrain) for N-tubulin in hairy1 TP-injected embryos.(B) The number of differentiating neurons (Myt1+ cells) is decreased upon injection of hairy1 TP.(C) Quantification of the Myt1+ cells in the forebrain (n = 7, p < 0.001) and the hindbrain (n = 7, p < 0.001). Myt1+ cells in the injected side were expressed as a percentage of the control side.(D) Hairy1 TP leads to forebrain-specific induction of apoptosis as indicated by TUNEL staining.(E) Quantification of the TUNEL-positive nuclei in the forebrain (n = 7, p < 0.001) and in the hindbrain (n = 5).(F) Immunostaining for pH3 in embryos injected in one side with hairy1 TP.(G) Relative number of pH3+ cells in the hairy1 TP-injected compared to the control side in the forebrain (n = 11) and the hindbrain (n = 9, p < 0.001).(H) In situ hybridization for N-tubulin (purple) in embryos electroporated in one side with hairy1 Δ3′UTR and lacZ DNA as a tracer. Light-blue staining indicates the electroporated area. op, olfactory placodes. Scale bars, 20 μm. In all panels, FITC was used to identify the MO-injected side; DNA was counterstained with DAPI; CNS tissue is outlined with a dashed line; and error bars represent SEM.
	Figure 7. Mechanism of miR-9 Function(A) Forebrain sections of miR-9 MO or hairy1 TP-injected embryos analyzed for CyclinD1, p27Xic1, and Fgf8 expression by in situ hybridization.(B) Hindbrain sections of miR-9 MO or hairy1 TP-injected embryos analyzed for CyclinD1, p27Xic1, Wnt1, and Zic1 expression.(C) Representative western blot for endogenous p53 protein levels in forebrain or hindbrain tissue isolated from X. tropicalis embryos injected with control MO, miR-9 MO, or hairy1 TP. Numbers represent the mean from three experiments, 60 embryos each.(D) Real-time PCR analysis for Mdm2 expression, normalized for the ribosomal protein RPL8 (n = 3 experiments, 20 embryos each). Error bars represent SEM.(E) Model for miR-9 function in cell survival and progenitor proliferation.

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Aruga, Zic1 promotes the expansion of dorsal neural progenitors in spinal cord by inhibiting neuronal differentiation. 2002, Pubmed